Apoptosis, or programmed cell death, is a principal mechanism by which organisms eliminate unwanted cells. The deregulation of apoptosis, either excessive apoptosis or the failure to undergo it, has been implicated in a number of diseases such as cancer, acute inflammatory and autoimmune disorders, ischemic diseases and certain neurodegenerative disorders (see generally Science, 1998, 281, 1283-1312; and Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).
Caspases are a family of cysteine protease enzymes that are key mediators in the signaling pathways for apoptosis and cell disassembly (Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathways vary depending on cell type and stimulus, but all apoptosis pathways appear to converge at a common effector pathway leading to proteolysis of key proteins. Caspases are involved in both the effector phase of the signaling pathway and further upstream at its initiation. The upstream caspases involved in initiation events become activated and in turn activate other caspases that are involved in the later phases of apoptosis.
Caspase-1, the first identified caspase, is also known as interleukin converting enzyme or “ICE.” Caspase-1 converts precursor interleukin-1β (“pIL-1β”) to the pro-inflammatory active form by specific cleavage of pIL-1β between Asp-116 and Ala-117. Besides caspase-1 there are also eleven other known human caspases, all of which cleave specifically at aspartyl residues. They are also observed to have stringent requirements for at least four amino acid residues on the N-terminal side of the cleavage site.
The caspases have been classified into three groups depending on the amino acid sequence that is preferred or primarily recognized. The group of caspases, which includes caspases 1, 4, and 5, has been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site. Another group which includes caspases 2, 3 and 7, recognizes aspartyl residues at both positions 1 and 4 on the N-terminal side of the cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10, tolerates many amino acids in the primary recognition sequence, but seems to prefer residues with branched, aliphatic side chains such as valine and leucine at position 4.
The caspases have also been grouped according to their perceived function. The first subfamily consists of caspases-1 (ICE), 4, and 5. These caspases have been shown to be involved in pro-inflammatory cytokine processing and therefore play an important role in inflammation. Caspase-1, the most studied enzyme of this class, activates the IL-1β precursor by proteolytic cleavage. This enzyme therefore plays a key role in the inflammatory response. Caspase-1 is also involved in the processing of interferon gamma inducing factor (IGIF or IL-18) which stimulates the production of interferon gamma, a key immunoregulator that modulates antigen presentation, T-cell activation and cell adhesion.
The remaining caspases make up the second and third subfamilies. These enzymes are of central importance in the intracellular signaling pathways leading to apoptosis. One subfamily consists of the enzymes involved in initiating events in the apoptotic pathway, including transduction of signals from the plasma membrane. Members of this subfamily include caspases-2, 8, 9 and 10. The other subfamily, consisting of the effector caspases 3, 6 and 7, is involved in the final downstream cleavage events that result in the systematic breakdown and death of the cell by apoptosis. Caspases involved in the upstream signal transduction activate the downstream caspases, which then disable DNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton and finally fragment the cell.
Knowledge of the four amino acid sequence primarily recognized by the caspases has been used to design caspase inhibitors. Reversible tetrapeptide inhibitors have been prepared having the structure CH3CO—[P4]-[P3]-[P2]—CH(R)CH2CO2H where P2 to P4 represent an optimal amino acid recognition sequence and R is an aldehyde, nitrile or ketone capable of binding to the caspase cysteine sulfhydryl. Rano and Thornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med. Chem. Lett. 3, 2689-2692 (1993); and Nicholson et al., Nature 376, 37-43 (1995). Irreversible inhibitors based on the analogous tetrapeptide recognition sequence have been prepared where R is an acyloxymethylketone (—COCH2OCOR′), wherein R′ is exemplified by an optionally substituted phenyl such as 2,6-dichlorobenzoyloxy, and where R is COCH2X wherein X is a leaving group such as F and Cl. Thornberry et al., Biochemistry 33, 3934 (1994); and Dolle et al., J Med. Chem. 37, 563-564 (1994).
The utility of caspase inhibitors to treat a variety of mammalian disease states associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors. For example, in rodent models, caspase inhibitors have been shown to reduce infarct size and inhibit cardiomyocyte apoptosis after myocardial infarction, to reduce lesion volume and neurological deficit resulting from stroke, to reduce post-traumatic apoptosis and neurological deficit in traumatic brain injury, to effectively treat fulminant liver destruction, and to improve survival after endotoxic shock. Yaoita et al., Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow and Metabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med., 184, 2067 (1996); and Grobmyer et al., Mol. Med., 5, 585 (1999).
In general, the peptidic inhibitors described above are very potent against some of the caspase enzymes. However, this potency has not always been reflected in cellular models of apoptosis. In addition peptide inhibitors are typically characterized by undesirable pharmacological properties such as poor oral absorption, poor stability and rapid metabolism. Plattner and Norbeck, in Drug Discovery Technologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England, 1990).
Recognizing the need to improve the pharmacological properties of the peptidic caspase inhibitors, peptidomimetic and non-natural amino acid peptide inhibitors have been reported.
WO 96/40647 discloses ICE inhibitors of the formula:
wherein B is H or an N-terminal blocking group; R1 is the amino acid side chain of the P1 amino acid residue wherein the P1 amino acid is Asp; Pn is an amino acid residue or a heterocyclic replacement of the amino acid wherein the heterocycle is defined in the application; R4 is hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl; m is 0 or a positive integer; and X is N, S, O, or CH2.
WO 97/22619 discloses inhibitors of interleukin-1β converting enzyme of the formula:
wherein X1 is —CH; g is 0 or 1; each J is independently selected from the group consisting of —H, —OH, and —F, provided that when a first and second J are bound to a C and said first J is —OH, said second J is —H; m is 0, 1, or 2; T is, inter alia, —CO2H; R1 is
where each Z is independently CO or SO2; R3 is as defined in the application; each X is independently selected from the group consisting of ═N— and ═CH—; and R20 is chosen from a group containing

WO 98/16502 discloses aspartate ester inhibitors of interleukin-1β converting enzyme of the formula:
wherein R1 is, inter alia, R5N(Ra)CHR6CO—; R2 is certain groups; R6 is H, C1-6 alkyl, —(CH2)naryl, —(CH2)nCO2Ra, hydroxyl substituted C1-6 alkyl, or imidazole substituted C1-6 alkyl; each Ra is independently hydrogen, C1-6 alkyl or (CH2)naryl; and R5 is, inter alia, CONRaRa.
WO 99/18781 discloses dipeptide apoptosis inhibitors having the formula:
where R1 is an N-terminal protecting group; AA is a residue of any natural α-amino acid, or β-amino acid; R2 is H or CH2R4 where R4 is an electronegative LG such as F, Cl, TsO—, MeO—, ArO—, ArCOO—, ArN— and ArS—; and R3 is alkyl or H.
WO 99/047154 discloses dipeptide apoptosis inhibitors having the formula:
where R1 is an N-terminal protecting group; AA is any non-natural amino acid or amino acid residue; and R2 is an optionally substituted alkyl or H as defined in the application.
WO 00/023421 discloses (substituted) acyl dipeptide apoptosis inhibitors having the formula:
where n is 0, 1, or 2; q is 1 or 2; A is a residue of any natural or non-natural amino acid; B is a hydrogen atom, a deuterium atom, Cl-10 straight chain or branched alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH2)mcycloalkyl, (CH2)mphenyl, (CH2)m(substituted phenyl), (CH2)m(1- or 2-naphthyl), (CH2)mheteroaryl, halomethyl, CO2R13, CONR14R15, CH2ZR16, CH2OCOaryl, CH2OCO(substituted aryl), CH2OCO(heteroaryl), CH2OCO(substituted heteroaryl), or CH2OPO(R17)R18, where R13, R14, R15, R16, R17, R18 and m are as defined in the application; R2 is selected from a group consisting of hydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, and (CH2)mNH2; R3 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; X is CH2, C═O, O, S, NH, C(═O) NH or CH2OCONH; and Z is an oxygen or a sulfur atom.
WO 00/061542 discloses dipeptide apoptosis inhibitors having the formula:
where R1 is an optionally substituted alkyl or hydrogen group; R2 is hydrogen or optionally substituted alkyl; Y is a residue of a natural or non-natural amino acid; R3 is an alkyl, saturated carbocyclic, partially saturated carbocyclic, aryl, saturated heterocyclic, partially saturated heterocyclic or heteroaryl group, wherein said group is optionally substituted; and X is O, S, NR4, or (CR4R5)n where R4 and R5 are, at each occurrence, independently selected from the group consisting of hydrogen, alkyl and cycloalkyl, and n is 0, 1, 2, or 3; or X is NR4, and R3 and R4 are taken together with the nitrogen atom to which they are attached to form a saturated heterocyclic, partially saturated heterocyclic or heteroaryl group, wherein said group is optionally substituted; or X is CR4R5, and R3 and R4 are taken together with the carbon atom to which they are attached to form a saturated carbocyclic, partially saturated carbocyclic, aryl, saturated heterocyclic, partially saturated heterocyclic or oxygen-containing heteroaryl group, wherein said group is optionally substituted; and provided that when X is O, then R3 is not unsubstituted benzyl or t-butyl; and when X is CH2, then R3 is not H.
Generally, the term tumor necrosis factor (TNF) refers to two closely related cytokines (encoded by separate genes) known as tumor necrosis factor-alpha (TNF, cachectin) and tumor necrosis factor-beta (lymphotoxin, TNF-beta). Both cytokines interact with the same cell membrane receptors, and both have been implicated as pathogenic mediators of human illness.
TNF-alpha participates in the signaling pathways that regulate cell apoptosis and inflammation. TNF-alpha is also known as TNFSF2, TNFA and DIF. TNF-alpha is a pro-inflammatory mammalian protein capable of inducing cellular effects by virtue of its interaction with specific cellular receptors. It is produced primarily by activated monocytes and macrophages. Lipopoly-sacccharide (LPS, also called endotoxin), derived from the cell wall of gram negative bacteria, is a potent stimulator of TNF-alpha synthesis.
Due to the deleterious effects which can result from an over-production or an unregulated production of TNF-alpha, considerable efforts have been made to regulate the serum level of TNF-alpha. The pathology of a number of diseases are affected by TNF-alpha, including, restinosis, inflammatory diseases of the central nervous system, demyelinating diseases of the nervous system, multiple sclerosis, septic arthritis, aneurysmal aortic disease, traumatic joint injury, peridontal disease, macular degeneration, diabetic retinopathy, occular inflammation, keratoconus, Sjogren's syndrome, corneal graft rejection, cachexia, and anorexia.
While a number of caspase and TNF-alpha inhibitors have been reported, it is not clear whether they possess the appropriate pharmacological properties to be therapeutically useful. Therefore, there is a continued need for small molecule caspase and TNF-alpha inhibitors that are potent, stable, and have good penetration through membranes to provide effective inhibition of apoptosis in vivo. Such compounds would be extremely useful in treating the aforementioned disease states where caspase enzymes and/or TNF-alpha cytokines play a role.